Granulocytes play an important role in host resistance to bacterial, fungal and parasitic pathogens. This resistance is effected through at least two different pathways. In the oxygen dependent pathway, the granulocytes produce toxic oxygen metabolites which participate in killing the invading pathogen. The second pathway is oxygen independent and consists of production and storage of proteins which have potent antimicrobial activity. The importance of these granulocytic defense mechanisms is indicated by the finding that neutropenic patients are unusually susceptible to long term infections, which are sometimes fatal. A number of microbicidal proteins and peptides have been demonstrated in granulocytes, including eosinophil major basic protein, eosinophil cationic protein, bacteria permeability increasing factor and a group of small antibiotic peptides termed defensins which have been isolated from humans (M. E. Selsted et al., J. Clin. Invest. 76: 1436 (1985)), rabbits (M. E. Selsted et al., J. Biol. Chem. 260: 4579 (1985)), and guinea pigs (M. E. Selsted et al., Infec. Immun. 55: 2281 (1987)).
The reported sequences of the known human, rabbit and guinea pig defensins are shown in FIG. 4. In addition, U.S. Pat. No. 4,543,252, issued Sep. 24, 1985 and assigned to the Regents of the University of California discloses cationic oligopeptides of up to about 35 amino acids which have microbicidal activity. A comparison of various defensin sequences show that six cysteines are completely conserved among human, rabbit, and guinea pig defensins. It is believed that these residues may be involved in maintenance of secondary structure. In addition, five other residues (2 arg, 2 gly, glu) are conserved in all human and rabbit defensins, three of which are also conserved in guinea pig defensin.
A single difference among human defensins at the N-terminus is sufficient to produce significant changes in microbicidal potency and selectivity (R. I. Lehrer et al., Infect. Immun 42: 10 (1983)). Moreover, the larger differences among the rabbit defensins drastically alter their bactericidal activity (M. E. Selsted et al., Infect. Immun. 45: 150 (1984)), their Candidacidal activity (M. E. Selsted et al Infect Immun. 49: 202 (1985)) and their tumor cell cytolytic activity (A. Lichtenstein, Blood 68: 1407 (1986)). Therefore, although sequence conservation suggests certain secondary structures may be necessary for activity, it is clear that these structures are not sufficient for activity and that the potency and the specificity of the peptides are almost solely determined by sequence in non-conserved regions.
The polypeptides of the subject invention shares the cysteine backbone of defensins, but diverges radically in its remaining sequence. It is different in 16 of positions from any of the human or rabbit defensins, and therefore would be expected to differ widely in activity. Moreover, 22 of 33 positions differ from described human defensins. Human defensins are likely to be more important therapeutically because non-human defensins would be expected to trigger undesirable immune responses.
When tested in vitro the purified polypeptide of this invention has been found to be significantly more active in microbicidal assays against Escherichia coli, Streptococcus faecalis and Candida albicans than a mixture of previously described defensins.
Finally, U.S. Ser. No. 125,684, filed Nov. 25, 1987, in the names of Joelle E. Gabay and Carl F. Nathan and assigned to Cornell Research Foundation and the Rockefeller University discloses a series of peaks obtained by reverse-phase, high pressure, liquid chromatography of an extract of human blood. Specifically, Peak 2 of FIG. 13 of U.S. Ser. No. 125,684 discloses the existence of the polypeptide of this invention. However, it is important to understand that the data presented in U.S. Ser. No. 125,684 is that of applicants on the subject application and that this application is assigned to the exclusive licensee of U.S. Ser. No. 125,684. Moreover, it is important to note that U.S. Ser. No. 125,684 does not disclose that Peak 2 of FIG. 13 which contains the polypeptide of this invention has antibacterial or antifungal activity and does not disclose the amino acid sequence or any other characterization of the polypeptide of Peak 2 of FIG. 13.